Stem cell-derived hepatocytes in co-culture and uses thereof

a technology of stem cells and hepatocytes, which is applied in the field of in vitro cultures of human hepatic cells (hepatocytes), can solve the problems of severely limited resources of phhs and substantially limit their use in toxicology screening

Active Publication Date: 2015-08-27
COLORADO STATE UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PHHs are a severely limited resource given shortages in donor livers, and their quality for in vitro use can vary considerably across different cell lots.
Current research indicates however that iHeps remain more fetal-like in phenotype, which substantially limits their use in toxicology screening.

Method used

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  • Stem cell-derived hepatocytes in co-culture and uses thereof
  • Stem cell-derived hepatocytes in co-culture and uses thereof
  • Stem cell-derived hepatocytes in co-culture and uses thereof

Examples

Experimental program
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Effect test

example 1

Micropatterned Pure iHep Cultures

[0099]Micropatterned cultures of primary iHeps (Cellular Dynamics International) were first prepared to confirm the ability of iHeps to exhibit characteristics of human hepatocytes. Initial iHep processing proceeded as follows. Induced pluripotent stem cell-derived human hepatocytes were provided fresh in suspension by Cellular Dynamics International (CDI). Using a published protocol, CDI generates ˜95% pure iHeps (via α1-antitrypsin). Upon arrival, iHeps were processed according to manufacturer-supplied protocols. Briefly, iHep cell aggregates were rinsed with divalent cation-free Hank's Balanced Saline Solution (HBSS, Hyclone), dissociated with 0.5% trypsin-EDTA (Life Technologies), neutralized with a 1:1 solution of Roswell Park Memorial Institute 1640 (RPMI, Life Technologies) media and fetal bovine serum (FBS, Life Technologies), and pelleted via centrifugation. iHeps were re-suspended in Kryothaw (SciKon Innovation, Inc.) and further centrifuge...

example 2

Micropatterned Co-Cultures of iHeps and iMPHs

[0103]Micropatterned co-cultures (iMPCCs) of iHeps and iMPHs were established using the following methods.

[0104]Murine embryonic 3T3-J2 fibroblasts were the gift of Howard Green (Harvard Medical School). Cells were cultured at 37° C., 10% CO2 in Dulbecco's Modified Eagle's Medium (DMEM) with high glucose, 10% (v / v) calf serum, and 1% (v / v) penicillin-streptomycin.

[0105]Tissue culture polystyrene 24-well and 96-well plates (BD Falcon) were homogeneously coated with 25 μg / mL rat tail collagen I (BD Biosciences) and subjected to soft lithography-based methods to micropattern circular collagenous islands (500 μm diameter with 1200 μm center-to-center spacing). To establish iMPCCs, iHeps were seeded at a density of 6.66×105 cells / mL into micropatterned wells (300 μL and 50 μL per well in 24-well and 96-well plates, respectively) and agitated every 20 minutes to distribute cells for selective binding to collagen islands. Following 4-5 h for com...

example 3

Qualitative Assessment of iMPCC Stability

[0107]To qualitatively assess iMPCC stability, iHep morphology was monitored using an EVOS®FL cell imaging system with standard 10× or 20× objectives and phase contrast. For immunofluorescence, DAPI (357 / 44 Ex, 447 / 60 Em), GFP (470 / 22 Ex, 510 / 42 Em), and RFP (531 / 40 Ex, 593 / 40 Em) LED light cubes were used.

[0108]For albumin immunofluorescence, live cultures were washed once in phosphate buffered saline (PBS), fixed in 4% paraformaldehyde (Alfa Aesar) for 15 minutes, and followed with 3×PBS rinses (5 minutes each). Fixed cells were permeabilized using 0.1% triton X-100 (Amresco) for 10 minutes followed by another 3×PBS rinses (5 minutes each). Samples were incubated at 37° C. for 30 minutes in blocking solution, consisting of 20% goat serum (Thermo Scientific) in PBS. Primary rabbit anti-human intracellular albumin antibody (Rockland) was added to blocking solution 1:100 and incubated for 1 hour at 37° C. After incubation, cultures were washed...

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Abstract

The present disclosure provides co-cultures of human pluripotent stem cell derived hepatocytes and at least one non-parenchymal cell population in vitro, methods of preparing the co-cultures and methods of using the co-cultures for high throughput screening and evaluation of drug candidates. The stem cell derived hepatocyte co-culture system provides an in vitro model in which cell viability and relatively mature hepatocyte phenotype of stem cell derived hepatocytes are maintained for extended periods relative to conventional monoculture.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Patent Application No. 61 / 927,285 filed Jan. 14, 2014, the entire disclosure of which is incorporated herein by reference.GOVERNMENT LICENSE RIGHTS[0002]This invention was made with government support under CBET-1351909 awarded by the National Science Foundation. The government has certain rights in the invention.FIELD OF THE INVENTION[0003]The disclosure relates to in vitro cultures of human hepatic cells (hepatocytes), is and in particular to co-cultures of stem cell derived human hepatocytes with stromal cells, and use of the co-cultures in drug screening.BACKGROUND OF THE INVENTION[0004]Due to significant species-specific differences in liver pathways, in vitro models of the human liver play an important role in drug development and mechanistic investigations. Isolated primary human hepatocytes (PHHs) are ideal for constructing such models because they can maintain high levels of...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/071G01N33/50
CPCC12N5/067C12N2502/13C12N2506/45G01N33/5067C12N2501/237C12N2501/39C12N2503/02C12N2533/90
Inventor KHETANI, SALMAN R.BERGER, DUSTIN R.WARE, BRENTON R.
Owner COLORADO STATE UNIVERSITY
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